1. Field of the Invention
The present invention relates to a process for inducing differentiation of an embryonic stem cell into a functional cell. More particularly, the present invention relates to a process for inducing differentiation of an embryonic stem cell into an ectodermal cell or an ectoderm-derived cell useful for cell medical treatment, the differentiation-induced cell and use thereof. Also, the present invention relates to a medium used in the above process, an antibody which specifically recognizes a stroma cell in the above process, an antigen recognized by the antibody and use thereof.
2. Brief Description of the Background Art
In general, an embryonic stem cell means a cell which can be cultured in vitro and can also differentiate into all cells including germ cells when injected into the vacuole of an embryo before implantation, such as blastocyst, of other individual, and is called an embryonic stem cell or an ES cell.
Relationship between the generation of the initial stage embryo and the embryonic stem cell is described below using mouse as an example.
While moving from the oviduct to the uterus, a mouse fertilized egg repeats its division into 2 cells, 4 cells and 8 cells, generates compaction in which adhesion among cells is increased when it becomes the 16-cell stage, and reaches the stage called morula where borders among cells become unclear. In addition, 3.5 days after fertilization, a space called blastcoel is formed inside the embryo and becomes blastocyst. The blastocyst of this stage comprises the outer trophectogerm layer and inner cell mass (ICM). The blastocyst is implanted onto the uterus wall spending at 4.5 to 5.5 days after fertilization. At the stage of implantation, surface cells facing the blastcoel in the inner cell mass are differentiated into primitive endoderm cells. A part of these cells separates from the embryo itself, migrates into inside of the trophectoderm layer and becomes parietal endoderm cells to form Reichert's membrane by secreting an extracellular matrix.
On the other hand, the primitive endodermal cells around the embryonic part form a cell layer called visceral endoderm. These parietal and visceral endoderms then become a supporting tissue for protecting the fetus itself and exchanging nourishment and waste matter between it and the mother body. Cells of the inner cell mass, which form the fetus body in the future, proliferate and form a cell layer called primitive ectoderm. The primitive ectoderm is also called embryonic ectoderm or epiblast. Since the embryo after implantation grows into a cylindrical form as a whole, the embryo after 5.5 to 7.5 days of implantation is called egg cylinder. In half of the base side of the egg cylinder to the uterus, an extraembryonic tissue which forms the placenta in the future is formed by differentiating from the trophectoderm. After 6.5 days of fertilization, a groove called primitive streak appears on the primitive ectoderm layer, and, in this part, the primitive ectoderm enters into a space between the primitive ectoderm layer and the visceral endoderm layer by changing to a mesenchymal cell-like form and migrates from the primitive streak toward all directions to form embryonic mesoderm. In this cell layer, cells which become the definitive endoderm of the fetus body in the future are also contained.
Thus, it is known that 3 germ layers of not only ectoderm but also mesoderm and endoderm of the fetus are produced from the primitive ectoderm, and that all tissues of the fetus are derived from the primitive ectoderm. Also, It has been found that cells of the nervous system and the epidermal system are formed from ectoderms, and the ectoderm destined to differentiate into nervous system cells is called neuroectoderm (neural ectoderm), and the ectoderm destined to differentiate into epidermal system cells is called non-neuroectoderm.
Among the cell lineage in the embryo generation process described above, individual blastomere staring from fertilized egg to morula, cells of the inner cell mass in the blastocyst and cells constituting the primitive ectoderm layer have a totipotency and have properties as undifferentiated embryonic stem cells. When a primitive ectoderm starts its differentiation into each germ layer, most of its cells lose the totipotency, but a part of them is left as a primordial germ cell which takes part in transmitting genes to the next generation. When the primitive ectoderm is differentiated into each germ layer, the primordial germ cell migrates in the rear together with the embryonic mesoderm layer invaginating from the primitive streak and appears in a specific region of the extraembryonic mesoderm at the base of allantois. The primordial germ cell then migrates toward the gonad primordium and forms an ovum or a spermatozoon according to the sexual differentiation of gonad.
The embryonic stem cell can be established by culturing the inner cell mass-constituting undifferentiated stem cell existing in the inside of blastocyst and frequently repeating dissociation and subculturing of the cell mass. It is known that the cell can repeat proliferation and subculture almost unlimitedly while maintaining its normal karyotype and has a pluripotency of differentiating into every type of cells just as the same as the inner cell mass.
When an embryonic stem cell is injected into the blastocyst of other individual, it is mixed with the cell of inner cell mass of the host embryo and forms a chimeric individual by contributing to the formation of embryo and fetus. In an extreme case, an individual fetus body mostly composed of the only embryonic stem cell injected can be produced. Among chimeric individuals, an individual in which the injected embryonic stem cell has contributed to the formation of a primordial germ cell which will produce an egg or a sperm in the future is called germ line chimera, and since an individual derived from the injected embryonic stem cell can be obtained by crossing the germ line chimera, it has been confirmed that the embryonic stem cell has a totipotency of differentiating into all cells (Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) (hereinafter referred to as “Manipulating the Mouse Embryo, A Laboratory Manual”); Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993) (hereinafter referred to as “Gene Targeting”); Biomanual Series 8, Gene Targeting, Production of Mutation Mouse Using ES Cell, Yodo-sha (1995) (hereinafter referred to as “Production of Mutation Mouse Using ES Cell”)).
When the inner cell mass of blastocyst is cultured like the usual primary culture, it directly differentiates into a fibroblast-like cell in most cases. In order to culture it while maintaining undifferentiated conditions, it is necessary in general to use a primary fibroblast cell produced from the fetus or STO cell derived from an SIHM mouse as a feeder cell (Gene Targeting, Production of Mutation Mouse Using ES Cell). By keeping an appropriate cell density on the feeder cell and repeating dissociation and subculture of the cell mass while frequently exchanging the culture medium, it becomes possible to maintain the conditions while keeping properties of the undifferentiated stem cell (Manipulating the Mouse Embryo, A Laboratory Manual).
As a factor for maintaining undifferentiated conditions of an embryonic stem cell, LIF (leukemia inhibitory factor) has been identified (A. G. Smith and M. L. Hooper, Dev. Biol., 121, 1 (1987); A. G. Smith et al., Nature, 336, 688 (1988); P. D. Rathjen et al., Genes Dev., 4, 2308 (1990)), and it has been reported that an embryonic stem cell having a totipotency can be isolated and cultured without using a feeder cell when LIF is added to the culture medium (J. Nichols et al., Development, 110, 1341 (1990); S. Pease et al., Dev. Biol., 141, 344 (1990)). Also, it has been shown that the addition of a family molecule of interleukin 6 sharing a subunit gp130 of LIF receptor as the common receptor is effective, instead of adding LIF itself to the culture medium (D. P. Gearing and G. Bruce, New Biol., 4, 61 (1992); J. I. Conover et al., Development, 119, 559 (1993); C. Piquet-Pellorce et al., Exp. Cell Res., 213, 340 (1994); D. Pennica et al., J. Biol. Chem., 270, 10915 (1995)).
In addition, since it has been reported that an embryonic stem cell capable of contributing to the formation of a germ line cell by maintaining undifferentiated conditions of the embryonic cell was established by jointly using interleukin 6 capable of directly activating gp130 and a soluble interleukin 6 receptor (K. Yoshida et al., Mech. Dev., 45, 163 (1994); J. Nichols et al., Exp. Cell Res., 215, 237 (1994); Japanese Published Unexamined Patent Application No. 51060/95, It has been found that intracellular signal transduction from gp130 is playing an important role in maintaining the pluripotency and undifferentiation of the embryonic stem cell. This is supported also by a fact that normal generation of initial stage embryo is observed in a deficiency mouse whose LIF gene and LIF receptor gene were destroyed using gene targeting techniques (C. L. Stewaet et al., Nature, 359, 76 (1992); J. L. Escary et al., Nature, 363, 361 (1993); M. Li et al., Nature, 378, 724 (1995); C. B. Ware et al., Development, 121, 1283 (1995)), but fetal death occurs during a period from the fetal age of 12.5 days to birth in a mouse whose gp130 gene was destroyed (K. Yoshida et al., Proc. Natl. Acad. Sci. USA, 93, 407 (1996)).
Since the first establishment of an embryonic stem cell in mice (M. J. Evans et al, Nature, 292, 154 (1981); G. R. Martin, Proc. Natl. Acad. Sci. USA, 78, 7634 (1981)), methods for establishing efficient embryonic stem cells such as methods for establishing embryonic stem cells in non-mice (U.S. Pat. No. 5,453,357; U.S. Pat. No. 5,670,372) have been studied, and embryonic stem cells have so far been established in rat (P. M. Iannaccone et al., Dev. Biol., 163, 288 (1994)), in domestic fowl (B. Pain et al., Development, 122, 2339 (1996); U.S. Pat. No. 5,340,740; U.S. Pat. No. 5,656,479)), in pig (M. B. Wheeler, Reprod. Fertil. Dev., 6, 563 (1994); H. Shim et al., Biol. Reprod., 57, 1089 (1997)), in monkey (J. A. Thomson et al., Proc. Natl. Acad. Sci. USA, 92, 7844 (1996)) and in human (J. A. Thomson et al., Science, 283, 1145 (1998); M. J. Shamblott et al., Proc. Natl. Acad. Sci. USA, 95, 13726 (1998)).
It is known that a teratoma in which various tissues are mixed is formed when an embryonic stem cell is transplanted, e.g., under the skin of an animal of the same line of the embryonic stem cell (Manipulating the Mouse Embryo, A Laboratory Manual).
Also, it has been reported that, in in vitro culturing, various cells such as endodermal cells, ectodermal cells, mesodermal cells, blood cells, endothelial cells, cartilage cells, skeletal muscle cells, smooth muscle cells, heart muscle cells, glial cells, nerve cells, epithelial cells, melanocytes and keratinocytes can be formed by inducing differentiation through the formation of a cell mass called embryoid body (hereinafter referred to as “EB”) in which embryonic stem cells are once aggregated to form a pseudo-embryonic state (P. D. Rathjen et al., Reprod. Fertil. Dev., 10, 31 (1998)). However, in the differentiation induction by this culturing method, spontaneous differentiation is generated by the formation of cell aggregation mass and, as a result, appearance of the intended cell is observed. Accordingly, it does not result in the efficient induction of a specified cell group and appearance of a variety of tissue cells is simultaneously observed.
Various attempts have been made for methods for efficiently inducing differentiation of nervous system cells from the embryonic stem cell.
It has been reported that expression of a transcription factor Pax3 and neurofilament important for the differentiation of nervous system cells is significantly increased when culturing of the stem cell after formation of EB is continued using a medium supplemented with NGF (nerve growth factor) on a glass dish coated with poly-L-lysine or laminin (G. Yamada et al., Biochem. Biophys. Res. Commun., 199, 552 (1994)). Based on the information that differentiation of an EC cell which will be described later into nervous system is accelerated by retinoic acid treatment (E. M. V. Jones-Villeneuve et al., J. Cell Biol., 94, 253 (1982); G. Bain et al., BioEssays, 16, 323 (1994)), its effect on embryonic stem cells has also been examined, and it has been reported that a neuron-like cell which generates action potential by developing axons appears at a high ratio of about 40%, when EB is cultured for 4 days in the presence of retinoic acid and then treated with trypsin to carry out monolayer culturing, and that expression of class III tubulin, neurofilament M subunit, GAP-43 (growth-associated protein-43) as a substrate of nerve-specific calmodulin binding kinase C, γ-aminobutyric acid (hereinafter referred to as “GABA”) receptor, NMDA (N-methyl-D-aspartate) receptor and synapsin is observed in this cell at a protein level, and expression of neurofilament L subunit, glutamic acid receptor, tyrosine hydroxylase, a transcription factor Brn-3, GFAP (glial fibrillary acidic protein) and a GABA synthesizing enzyme GAD (glutamic acid decarboxylase) is observed at a mRNA level (G. Bain et al., Dev. Biol., 168, 342 (1995); F. A. Michael et al., J. Neurosci., 16, 1056 (1996)).
Since it is known that Brn-3 is expressed in central nervous system (X. He et al., Nature, 340, 35 (1989)), and GAP-43 is expressed in nerve axon (L. I. Benowitz and A. Routtenberg, Trends Neurosci., 20, 84 (1997)), MAP-2 is expressed in nerve dendrite (L. I. Binder et al., Ann. NY Acad. Sci., 76, 145 (1986)), GFAP is expressed in glial cell (A. Bignami et al., Brain Res., 43, 429 (1972)), GABA receptor and GAD are expressed in inhibitory nerve (Y. Chang and D. I. Gottlieb, J. Neurosci., 8, 2123 (1988)) and glutamic acid receptor and NMDA receptor are expressed in excitatory, nerve, it is shown that signals of differentiation into various nervous system cells are simultaneously transmitted when the differentiation is induced using retinoic acid.
Also, it has been reported that differentiating induction to nervous cells was not observed when retinoic acid was simply allowed to react directly with embryonic stem cells without mediating the interaction of cells by EB formation (H. G. Slager et al., Dev. Gen., 14, 212 (1993)). It has been reported that, when 10−7 mol/l retinoic acid was allowed to react with monolayer-cultured embryonic stem cells, expression of GAP-43 was observed in about 50% of the cells 3 days thereafter, and expression of neurofilament-165 (S. H. Yen and K. L. Fields, J. Cell Biol., 88, 115 (1981)) in less than 5% of the cells 4 to 5 days thereafter, both at protein level, but most of the GAP-43 positive cells showed an endodermal cell-like form (W. G. van Inzen et al., Biochim. Biophys. Acta., 1312, 21 (1996)). It has been reported that a part of the GAP-43 positive cells show a glial cell-like morphology and about half thereof are neurofilament-165 positive cells, but both of the GAP-43 and neurofilament-165 have lower staining degree by antibody staining than the nervous cells induced by retinoic acid treatment after EB formation (W. G. van Inzen et al., Biochim. Biophys. Acta., 1312, 21 (1996)). Thus, it has been confirmed that the interaction among cells by EB formation is necessary for the efficient differentiation induction of nervous system cells.
In addition, it has been reported that, when action potential of the cells having glial cell-like morphology was measured using a patch clamp method, generation of the potential by 5-HT (5-hydroxytryptamin)-, GABA-, kainic acid-, glutamic acid-, dopamine- or carbachol-stimulation was observed in about half of the examined cells, but generation of action potential by carbachol-stimulation was not observed in the neuron-like cells induced by retinoic acid treatment after EB formation, used as a control, instead, generation of action potential by noradrenaline-stimulation was observed, thus showing that the interaction among cells by EB formation is also important for the determination of the direction of differentiation of nerve cells (W. G. van Inzen et al., Biochim. Biophys. Acta., 1312, 21 (1996)). It is known that the cell layer on the EB surface differentiates into a primitive endoderm-like form in the EB formation by cell aggregation and it is considered that the differentiation is induced by a certain interaction between the cell layer and inner undifferentiated cells, but its factor has not specifically been identified (P. D. Tathjen et al., Reprod. Fertil. Dev., 10, 31 (1998)).
Thereafter, as a result of further detailed analysis of the effect of retinoic acid on embryonic stem cells, it has been found that, when EB formed in a medium supplemented with retinoic acid is cultured in a dish for tissue culture, a nestin-positive precursor cell common for neuron and glial cells firstly appears, and then cells differentiated into GABAergic nerve cells, cholinergic nerve cells, GFAP positive astrocytes, and O4 positive (M. Schachner et al., Dev. Biol., 83, 328 (1981)) oligodendrocytes appear (A. Fraichard et al., J. Cell Sci., 108, 3181 (1995)).
Differentiation of neuron and glial cells from nestin-positive common precursor cells in the living body has been suggested by a labeling test using retrovirus (U. Lendahl et al., Cell, 60, 585 (1990); J. Price et al., Development Supplement, 2, 23 (1991); J. Price et al., Brain Pathol., 2, 23 (1992)), and then confirmed by the isolation of a precursor cell existing in the brain of the living body as a nervous system stem cell (S. J. Morrison et al., Cell, 88, 287 (1997); R. D. G. McKay, Science, 276, 66 (1997)).
However, when retinoic acid is used for the differentiation induction of an embryonic stem cell, it is used at a markedly higher concentration (10 to 100 times) than the physiologically existing concentration. Since the use of retinoic acid at a concentration higher than the physiologically existing concentration is disliked from the toxicity point of view, it is difficult to use the obtained cell in transplantation. Accordingly, attempts have been made to induce an embryonic stem cell into a nervous system cell under conditions more close to the physiological conditions without using retinoic acid.
The following has been reported. A nestin-positive and fatty acid binding protein (which is expressed in the brain)-positive (A. Kurtz et al., Development, 120, 2637 (1994)) nerve epithelial cell-like precursor cell (neuroepithelial precursor cell) is induced, when EB formed by 4 days of suspension culturing is adhered onto a tissue culture dish by 1 day of culturing and then cultured for 5 to 7 days using an ITSFn medium comprising insulin, transferrin, selenium chloride and fibronectin (A. Rizzino and C. Growley, Proc. Natl. Acad. Sci. USA, 77, 457 (1980)), and the precursor cell grows keeping as the precursor cell when cultured using an mN3 serum-free medium comprising bFGF (basic fibroblast growth factor) and laminin, but it differentiates into a central nervous system cell and a glial cell when cultured using the medium from which bFGF is removed, and synaptogenesis of excitatory nervous system and inhibitory nervous system is observed when culturing is continued using a serum-supplemented medium (S. Okabe et al., Mech. Dev., 59, 89 (1996)).
A possibility for the nervous system cell induced in vitro in this manner to function normally in the living body has also been examined.
It has been observed that when the mouse epithelial cell-like precursor cell induced using the ITSFn medium is transplanted into the cerebral ventricle of a rat of 16 to 18 days of fetal age, the transplanted precursor cell migrates to be incorporated by the brain tissue and differentiates into a nerve cell, an astrocyte and an oligodendrocyte, but they cannot be distinguished from the host cell morphologically (O. Brustle et al., Proc. Natl. Acad. Sci. USA, 94, 14809 (1997)). However, formation of teratoma tissues which are not observed in the original tissue is observed in the transplanted region, such as formation of a neural tube-like structural body actively repeating cell division and a small cluster of alkaline phosphatase positive undifferentiated cells.
Formation of such teratoma tissues has also been observed in the transplantation of a nervous system precursor cell induced from embryonic stem cell using retinoic acid (J. Dinsmore et al., Cell, Transplant., 5, 131 (1996); T. Deacon et al., Exp. Neurol., 149, 28 (1998)).
Thereafter, it has been reported that repair of myelin sheath was observed without forming teratoma, when a precursor cell of a glial cell was induced from embryonic stem cells and the glial precursor cell was transplanted into the brain or spinal cord of a rat congenitally lacking myelin sheath (O. Brustle et al., Science, 285, 754 (1999)). In this transplantation, a further differentiated glial cell precursor cell was induced from the above-mentioned nerve epithelial cell-like precursor cell induced using an ITSFn medium after the EB formation and used in the transplantation.
That is, it is shown that the cell differentiation-induced in this manner can be used in the transplantation, because differentiation into a glial precursor cell can be induced by culturing the induced nerve epithelial cell-like precursor cell for 5 days on a dish coated with polyornithine in a medium containing insulin, transferrin, progesterone, putrescine, selenium chloride, FGF2 (fibroblast growth factor 2) and laminin, pealing the cells using Hanks' buffer which does not contain calcium and magnesium, subculturing the cells at a cell density of ⅕ in a medium containing FGF2 and EGF (epidermal growth factor) and then, when the cells reached confluent, continuing the subculture at a cell density of ⅕ in a medium comprising FGF2 and PDGF-AA (platelet-derived growth factor-AA). It has been found that the cell differentiation-induced in this manner is a glial precursor cell, because it is A2B5-positive (M. C. Raff et al., Nature, 303, 390 (1983)) and its differentiation into an astrocyte and an oligodendrocyte is observed in vitro when cultured using a medium which does not comprise FGF2 and EGF.
Regarding cells having functions similar to the embryonic stem cell, their relationships with the embryonic stem cell are described below.
Various embryonal carcinoma cells (EC cells) have been established from a malignant teratoma (teratocarcinoma), as cell lines having a pluripotency like the case of an embryonic stem cell (M. J. Evans, J. Embryol. Exp. Morph., 28, 163 (1972)).
These cells are considered to be cells having the properties of an embryonic stem cell as an undifferentiated stem cell, because they express a gene to be used as a marker of an embryonic stem cell (E. G. Bernstine et al., Proc. Natl. Acad. Sci. USA, 70, 3899 (1973); S. B. Diwan and L. C. Steven, J. Natl. Cancer Inst., 57, 937 (1976); D. Solter and B. B. Knowles, Proc. Natl. Acad. Sci. USA, 75, 5565 (1978); B. A. Hosler et al., Mol. Cell. Biol., 9, 5623 (1989); S. C. Pruitt, Development, 120, 37 (1994)), they are capable of differentiating into various cells in vitro (G. R. Martin and M. J. Evans, Cell, 6, 467 (1975); G. R. Martin and M. J. Evans, Proc. Natl. Acad. Sci. USA, 72, 1441 (1975); M. W. McBurney, J. Cell. Physiol., 89, 441 (1976)), teratoma is formed from various tissues by their transplantation into congenic individuals (L. J. Kleinsmith and G. B. Pierce, Cancer Res., 24, 797 (1964)), they form chimeric individuals by contributing to fetus formation when injected into a blastocyst (B. Mintz and K. Illmensee, Proc. Natl. Acad. Sci. USA, 72, 3538 (1975); V. E. Papaioannou et al., Nature, 258, 70 (1975); M. J. Dewey et al., Proc. Natl. Acad. Sci. USA, 74, 5564 (1977)) and, although it is extremely rare, an example is reported on an embryonal carcinoma cell line capable of producing a germ line chimera (T. A. Stewart and B. Mintz, Proc. Natl. Acad. Sci. USA, 78, 7634 (1981)).
Also, it was shown that a cell line of a cell analogous to an embryonic stem cell appeared when bFGF was added in culturing a primordial germ cell, and was established as an EG cell (embryonic germ cell) (Y. Matsui et al., Cell, 70, 841 (1992); J. L. Resnic et al., Nature, 359, 550 (1992)). It has been found that this EG cell is capable of contributing to the formation of a germ line chimera (C. L. Stewart et al., Devel. Biol., 161, 626 (1994); P. A. Labosky et al., Development, 120, 3197 (1994)) and has the properties as the undifferentiated stem cell possessed by the embryonic stem cell. Since undifferentiated stem cells and germ cells have fairly common properties, it is considered that they can be mutually converted relatively easily, by changes in the controlling conditions of proliferation and differentiation.
On the other hand, with the advance in developmental engineering, possibility of preparing an embryonic stem cell of individual human has been reported. Since the creation of a sheep, Dolly, as a somatic cell nucleus-derived clone individual for the first time in an mammal by Wilmut et al. in 1997 (Wilmut et al., Nature, 385, 810 (1997)), creation of a cloned calf using the nucleus of a fetal cell (J. B. Cibelli et al., Science, 280, 1256 (1998)), a cloned calf using the nucleus of a skin, muscle, ear capsule, oviduct or proligerous cumulus cell (A. Iritani, Protein, Nucleic Acid and Enzyme, 44, 892 (1999)), a cloned goat (A. Baguisi et al., Nature Biotechnology, 17, 456 (1999)), a cloned mouse using the nucleus of proligerous cumulus cell (T. Wakayama et al., Nature, 394, 369 (1998)), a cloned mouse using a cell of male tail (T. Wakayama et al., Nature Genetics, 22, 127 (1999)) and a cloned mouse using the nucleus of embryonic stem cell (T. Wakayama et al., Proc. Natl. Acad. Sci. USA, 96, 14984 (1999); W. M. Rideout III et al., Nature Genetics, 24, 109 (2000)) has been reported, thus showing a possibility of creating cloned individuals of mammals by introducing the nucleus of a somatic cell into enucleated oocytes. Since it is possible to prepare an embryonic stem cell of individual human by combining this nucleus transplantation technique with a technique for establishing the embryonic stem cell, a possibility of applying it to organ plantation as a cell medical treatment has been pointed out (R. P. Lanza et al., Nature Medicine, 5, 975 (1999)). Also, it has been pointed out that it is possible to carry out more effective gene therapy by applying gene manipulation to an embryonic stem cell and to modify histocompatibility antigens (P. D. Rathjen et al., Reprod. Fertil. Dev., 10, 31 (1998)).
Next, effectiveness of the cell medical treatment in organ transplantation is described with examples.
Parkinson disease is a chronic progressive disease mainly caused by the degeneration of dopaminergic neurons of substantia nigra corpus striatum. A perlingual therapy mainly using L-DOPA (L-dihydroxyphenylalanine) has conventionally been carried out, but since it is necessary to carry out its internal use for a prolonged period of time, its effect gradually attenuates in many patients who then will suffer from side effects such as wearing off phenomenon, dyskinesia and the like. Accordingly, development of more effective therapeutic methods has been attempted, and a treatment for transplanting an abortion fetal brain to patients of Parkinson disease has been started. In the whole world, several hundred cases of abortion fetal brain transplantation treatment have so far been carried out. Recently, a double blindfold test on the transplantation of abortion fetal brain cells was carried out in the United States for 40 patients of Parkinson disease, and its usefulness was confirmed. In addition, a case has been reported in which the transplanted cell was fixed for 10 years or more and the transplanted cell formed a synapse with corpus striatum in some patients who underwent such an abortion fetal brain cell transplantation. Thus, it has been understood that the cell treatment for transplanting the brain of abortion fetus shows high efficiency for Parkinson disease, but a protest against the use of abortion fetuses is strong due to ethical problems. In addition, since close to 10 fetuses are practically required for the treatment of one patient, it meets with a great obstacle for its realistic application to the therapy. Accordingly, concern has been directed toward the development of a method for preparing a dopaminergic neuron in a large amount by a commonly acceptable method.
In view of these backgrounds, development of a method for inducing differentiation of a target functional cell selectively and efficiently from an undifferentiated stem cell which can be cultured while maintaining its pluripotency has been drawing attention, and various attempts have been made thereon. However, development of a method for efficiently inducing differentiation of a cell group without accompanying formation of teratoma is not successful yet in many functional cells. Also, induction of a target functional cell under an artificially controlled physiological environment, such as culture conditions which do not use serum or retinoic acid, is desired from the viewpoint of cell medical treatment, but such a method is not known. Particularly, a method for obtaining an ectoderm-derived cell, specifically a dopaminergic neuron having normal functions, by efficient differentiation induction from an undifferentiated stem cell is important and desired from the viewpoint of the medical treatment of patients of brain diseases including Parkinson disease, but such a method has not been developed yet.